The Effects of Sanitizers on Calonectria pseudonaviculata and C. henricotiae Conidia and Microsclerotia Viability1
The boxwood blight pathogens Calonectria pseudonaviculata (Cps), and C. henricotiae (Che), produce microsclerotia (ms) on and in leaf tissue and conidia in sticky masses that can be spread by equipment and tools. We evaluated the effects of sanitizers on conidia, excised ms or all Cps life stages in 4-mm-d leaf disks. Leaf disks and excised ms were exposed to sanitizers or water for 3 to 180 min (disks), or 0.5 to 30 min (ms), wicked dry and placed onto half-strength potato dextrose agar (PDA) to determine viability. Conidia were loaded onto a filter, exposed to alcohol concentrations for between 2 sec to 2 min and rinsed with water. Filters were backwashed with air to transfer conidia onto water agar and the percent germination was counted after 24 h. Cps in leaf disks was killed within 10 – 12 min for 70% ethanol and between 2 – 3 h for 0.525% NaOCl. Chlorophenol did not eliminate Cps from tissue. Individual ms were killed by 70% ethanol in 4 min. However, ms exposed to 0.525% NaOCl or 0.05% chlorophenol for 30 min retained viability. Conidia mortality of Cps and Che exposed to 10, 25, 40 and 70% ethanol was strongly influenced by concentration and duration of exposure. Most importantly, conidia of Cps and Che died after a 5 sec exposure to either 70% ethanol or isopropanol, and there were no differences between alcohols. Ethanol and isopropanol can quickly and inexpensively disinfest tools and contaminated surfaces. Species used in this study: Boxwood (Buxus × ‘Green Velvet’); Calonectria henricotiae. Gehesquière, Heungens and J.A. Crouch; C. pseudonaviculata (Crous et al.) L. Lombard et al. Chemicals used in this study: Ethanol; isopropyl alcohol; sodium hypochlorite (bleach); Chlorophenol (Lysol®); Polyethylene glycol sorbitan monolaurate (Tween 20).Abstract
Significance to the Horticulture Industry
Boxwood (Buxus sp.) is a popular ornamental used in historic and home landscapes as hedges, specimen plants and topiary, while cut greenery is sold as wreaths. Despite the current threat posed by boxwood blight disease, which first appeared in the U.S. in 2011, boxwood’s popularity continues to grow. Annual sales of boxwood in the U.S. before the introduction of the pathogen (in 2009) were $102.9 million; in 2014, when boxwood blight was reported from 18 states, they were approximately $126 million (Hall et al. 2021) and were part of the $46 million dollars in sales for cut greenery in 2015 (USDA National Agricultural Statistics Service 2017). In 2019, when boxwood blight was reported from 30 states (Daughtrey 2019), boxwood sales were $140.9 million but the top boxwood-growing states have shifted as the pathogen has spread (Hall et al. 2021). To ensure that boxwood continues to be a mainstay of landscapes, methods need to be developed to ensure that spread of the boxwood blight pathogen can be minimized.
Introduction
Boxwood blight, caused by Calonectria pseudonaviculata (Crous et al.) L. Lombard et al. (Cps) was first discovered in 2011 in Connecticut and North Carolina (Ivors et al. 2012). A second species, Calonectria henricotiae Gehesquière, Heungens and J.A. Crouch (Che), is present in Europe but not yet reported in the US. Both pathogens cause leaf and stem lesions and can quickly defoliate plants under conducive environmental conditions.
Long-distance spread of the pathogen is largely through movement of infected nursery plants, but short distance dispersal of the sticky spore masses produced on infected leaves and stems can be through irrigation runoff and rain splash, or transfer via equipment, clothing, and the fur or feathers of animals (Daughtrey 2019, Ruhl et al. 2018). The spores do not spread by wind when dry (LaMondia and Maurer 2020). Of special concern is spread during pruning and landscape maintenance, when pruning tools can become contaminated with spores (Bordas et al. 2016, Bush et al. 2016a, 2016b, Dart et al. 2016). These spores, however, are delicate and do not live long in soil (Dart et al. 2015). Spread may also occur through the movement of infected tissues containing microsclerotia (ms). Microsclerotia are persistent structures that can survive for long periods in litter and soil (Dart et al. 2015, Shishkoff and Camp 2016). Diseased leaves can be spread locally after adhering to lawn maintenance equipment or by being blown in the wind.
To prevent dissemination of the pathogen, therefore, it is necessary to sanitize for spores, microsclerotia and plant litter. Appropriate washing and sanitizing of tools, clothing and equipment are obvious parts of a best management practices system. In this paper we examine several disinfestants that might be used to clean tools and nursery surfaces: alcohols (ethanol and isopropanol), bleach (sodium hypochlorite, NaOCL), and chlorophenol (o-benzyl-p-chlorophenol, the active ingredient in some formulations of Lysol® disinfectant). As alcohol was most effective for killing conidia, microsclerotia, and all stages present in leaf tissue including hyphae and chlamydospores, we further examined ethanol and isopropanol to determine the efficacy of different time and concentration combinations against conidial viability, to provide information useful for practitioners to disinfest tools used for maintaining boxwood shrubs.
Of the alcohols used as antiseptics, ethyl alcohol, isopropyl alcohol and n-propanol are the most widely used. Although ancient medical knowledge was often trial-and-error rather than controlled experimentation, Hippocrates observed that wine could be used to clean wounds, and Theodoric of Cervia, citing his teacher, Hugo of Lucca, documented in 1267 the use of hot wine as a wound treatment (Cope, 1958) although it is unclear whether its antiseptic qualities were primarily due to the alcohol content. In the late 17th century, William Croone and Robert Boyle preserved biological specimens in alcohol (Bauer and Wahlgren 2013, Boyle 1666), although they didn’t understand the mechanism. With new understanding that disease was caused by microorganisms, Robert Koch tested several chemicals, including alcohols, for anti-bacterial properties (Koch 1881). In 1884, L.O. Buchholtz studied the effects of ethanol on microorganisms in a systematic fashion (Thofern 1982). Even today, little is known about the mode of action of alcohols, but because of the reported increased efficacy of 70% ethanol in killing bacteria compared to 95% (Kruse et al. 1963), it is generally supposed that alcohols in the presence of water cause membrane damage and rapid denaturation of proteins, with subsequent interference with metabolism and cell lysis (McDonnell and Russell 1999, Rutala et al. 2008).
Antoine-Germain Labarraque recognized the antibiotic properties of hypochlorite in 1821 (Thofern 1982). In 1894, Moritz Traube suggested the chlorination of drinking water and in 1897 bleach was used against typhus (Traube 1894, Thofern 1982). Household bleach, in aqueous solutions of 5.25 – 8.25% sodium hypochlorite, is the most common hypochlorite used as a disinfectant/disinfestant. Rutala et al. (2008) considered hypochlorites to have a broad spectrum of antimicrobial activity, leaving no toxic residues, unaffected by water hardness, inexpensive and fast acting, with no unreasonable adverse effects to the environment when correctly used. Disadvantages include corrosiveness to metals, inactivation by organic matter, discoloring of fabrics, and release of toxic chlorine gas when mixed with ammonia or acid. Rutala et al. did not provide a mode of action for free chlorine but suggested that it might consist of some combination of oxidation of sulfhydryl enzymes and amino acids, ring chlorination of amino acids, loss of intracellular contents, decreased uptake of nutrients, inhibition of protein synthesis, decreased oxygen uptake, oxidation of respiratory components, decreased adenosine triphosphate production, breaks in DNA, and depressed DNA synthesis. They cited many instances of effective inactivation of human pathogens in seconds or minutes of exposure.
Phenol was isolated from coal tar by Friedlieb Ferdinand Runge in 1834 and first recommended as a disinfectant in 1861 by Lemaire, although the first systematic attempts to prove its antimicrobial effects were done by Lister in his pioneering work on antiseptic surgery in 1867 (Thofern 1982). In the past 30 years, however, antimicrobial research has concentrated on phenol derivatives. The mode of action of phenol at high concentrations is as a protoplasmic poison; lower concentrations cause bacterial death by inactivation of essential enzyme systems and leakage of essential metabolites from the cell wall (Rutala et al. 2008).
Rutala et al. (2008) thought the ideal disinfestant should be broad spectrum, fast acting, unaffected by environmental factors, water-soluble and compatible with soaps and detergents, nontoxic, compatible with surfaces and tools, and should not cause the deterioration of cloth, rubber, plastics, and other materials. It should have a residual effect on treated surfaces, be easy to use, have no strong odor, and be economical, stable and environmentally friendly. They further said that efficacy depended on the number of organisms and whether they clumped, the location of microorganisms (crevices being more difficult to disinfest than flat surfaces), the nature of the organism (bacterial spores being most resistant), physical and chemical factors (temperature, pH, relative humidity, and water hardness), the presence of organic and inorganic matter (interference by chemical reaction or as a physical barrier), and duration of exposure, which we address here. The duration of exposure must be long enough to effectively sterilize tools and equipment but short enough to be efficient for use by growers and landscapers.
Material and Methods
Leaf disc preparation
Infection of plants. Newly expanded leaves of one-year-old Buxus × ‘Green Velvet’ were inoculated with approximately 200 conidia of Cps-CT1 in a single drop and incubated in the greenhouse for 7 days until symptomatic leaves were heavily infected, when leaf discs were prepared.
Prepared sanitizers.
The treatments were prepared in this manner: 1) a commercial 95% v/v ethanol product was used, and in in these experiments was diluted to 70%, 2) a commercial NaOCl product (bleach containing 5.25% w/v NaOCl) diluted to 10% bleach (0.525% NaOCl), 3) a commercial product (Lysol containing 5% w/v chlorophenol) diluted to 43 ppm chlorophenol (a high experimental rate) and 5.37 ppm chlorophenol (a label rate). Leaf discs were cut from infected B. × ‘Green Velvet’ using a size 1 cork borer (4 mm diam) and 3 – 4 discs were added to small beakers containing 10 mL of each sanitizer. Discs were removed at specified times (3 – 15 min for ethanol, 30 – 180 min for NaOCl and chlorophenol; see Fig. 1), blotted, then placed onto Petri plates of half-strength PDA (BD Difco™ Difco, Becton, Dickenson and Company, 7 Loveton Circle, Sparks MD 21152). Growth of the pathogen out of the leaf discs was evaluated after ~1 wk to determine effectiveness of sanitizers. The procedure was repeated three times.
Citation: Journal of Environmental Horticulture 43, 2; 10.24266/0738-2898-43.2.83
Cps microsclerotia procedure
Prepared sanitizers.
Products used were 0.525% NaOCl, 70% ethanol, and chlorophenol solutions at 5.37 and 43 ppm, as defined earlier.
Individual microsclerotia (multiple isolates of Cps [Cps-CT1, -CT2 and -CT3]) were carefully lifted out of 1/2 PDA cultures with fine tweezers and rinsed with tap water over a #80 (180µ) sieve to remove any associated media, then exposed to NaOCL, ethanol or chlorophenol for 5, 15 or 30 min, rinsed in sterile distilled water (sdH20) and plated to rate growth. This experiment was repeated three times. Additional ethanol trials were performed with shorter intervals: 30 sec, 1 min, 2 min, and 4 min.
Cps conidia procedure for testing ethanol, chlorophenol and NaOCl
Prepared sanitizers.
Products used were ethanol (14 and 70%), NaOCl (0.105 and 0.525%), and chlorophenol (1.07 and 5.37 ppm), each prepared with 1 drop of Tween per 100 mL of sdH2O.
Conidia of Cps-CT1 were rinsed from one-week-old half-strength PDA plates and diluted to a concentration of approximately 1.0 × 105 per mL. Drops of conidial suspension were placed on a plate of half-strength PDA to test germination. For each treatment, 1.5 mL of conidial suspension was added to a 2 mL microcentrifuge tube and centrifuged for 1 min at 12,000 rpm. Excess liquid was removed from centrifuge tubes without disturbing the pellet, and that volume of liquid was replaced with either the full-strength sterilant or its 1:5 dilution. Each tube was then shaken for 30 sec. Tubes were then centrifuged again for 1 min. This procedure was also used for controls (200 ml sdH2O containing 2 drops of Tween 20, henceforth called “tween water”). Treated pellets were rinsed in tween water three times (each time shaken and re-pelleted) and after the last rinse, sdH2O was added, the tube shaken to resuspend the spores, and three drops were placed on a plate of half-strength PDA to evaluate conidial germination (out of 100 counted spores, with six replicates).
Cps conidia procedure for testing ethanol and isopropanol at shorter exposures
Prepared sanitizer.
Products used were commercial 99% w/v preparations of ethanol and isopropanol diluted to 10, 15, 22, 40 and 50%.
A sterile cellulosic syringe filter (0.22 µm) (Cameo 25ES, Micron Separations, Inc., Westboro, MA 01581) was placed in a micropipette tip and loaded with 0.5 mL of a conidial suspension (1.5 × 104 – 1.0 × 105 conidia per mL). To that was added 0.5 ml of the appropriate alcohol treatment and after the specified time (Table 4) rinsed with 1 mL sdH2O, then the filter was backwashed with air to create a drop containing conidia that had been exposed to alcohol on a water agar plate. After 24 h, the number of germinating conidia were counted for the first 100 conidia observed at 200 – 400× magnification; the experiment was replicated five times.
Ethanol and isopropanol effects against conidia of Cps and Che
Calonectria isolates.
For ethanol tests, we used Calonectria pseudonaviculata CT-1, an isolate collected in Connecticut, and C. henricotiae JKI 2106 from Europe. For isopropanol tests, six isolates were used: three Cps (BD6c, CT-1, and T) and three Che (JKI 2106, 78-che, and 182-che). These isolates were previously used to evaluate heat sensitivity in conidia and ms (Miller et al. 2018; Shishkoff et al. 2021).
Prepared sanitizers.
Ethanol at 10, 25, 40 and 70%; isopropanol at 70%.
To determine ethanol’s effect on viability of Calonectria conidia for both Cps and Che, experiments varied the percent ethanol content as well as duration of exposure. An experiment consisted of testing one fungal isolate at one ethanol concentration for different durations of exposures. For each experiment, spores were adjusted to a concentration of approximately 1 × 102 spores per mL in sdH2O. A ten mL aliquot of the spore suspension was passed through a 0.45 µm Millipore filter (MilliporeSigma, 3050 Spruce St., St. Louis, MO 63103) attached to a Buchner funnel under vacuum. The water from the spore suspension passed through the filter, leaving a layer of spores deposited on the upper surface of the filter. A given volume of ethanol solution (0, 5, 10 15, 20 and in some experiments 25 mL) was randomly selected and passed through the filter under vacuum while the exposure time was recorded using a stopwatch to measure the elapsed time. The instant the last of the ethanol solution had passed through the filter, 15 mL of sdH2O was added to the filter apparatus to stop the exposure to ethanol. When the water had passed through the filter, the filter was removed and placed firmly face down on the surface of a plate of GYT agar (Glucose Yeast-extract Tyrosine agar media, prepared (per liter distilled water) with 30 g Glucose, 200 mg yeast extract, 80 mg tyrosine, and 20 g Difco Agar [Hunter 1992]). The plate was labeled with the isolate, percent strength of the ethanol solution, and the duration of exposure. The filter apparatus was sprayed internally with ethanol under vacuum and rinsed with sdH2O under vacuum before being used again. This procedure continued until two or three replicates of each volume of ethanol had been used. For “0” exposure samples, spores deposited on the filter paper were immediately treated with the 15 ml of sdH2O before being placed on agar. After all replicates were processed, the filter was carefully removed from the surface of each agar plate and the plate examined to make sure that hundreds of spores had been displaced from the filter to the agar surface. The plates were stored at room temperature overnight and then examined under a dissecting microscope approximately 24 h later. One hundred randomly selected spores were examined for each plate, and the percentage germination of spores recorded. This experiment was repeated for 70% isopropanol using three isolates of Cps and three of Che. The isopropanol experiment was run twice for each isolate.
Statistics.
Percent survival values from the leaf disk and microsclerotia experiments were subjected to regression analysis of survival vs. duration of exposure. Where regressions were not significant, the statistical significance for individual time points were investigated. These binomial data (survival or not) for treatment and the water control were compared using the total survival and non-survival events via Fisher’s Exact test (Lowry 2023).
Tests comparing ethanol and isopropanol as disinfectants or disinfestants for killing conidia were analyzed with non-transformed proportion mortality and with this proportion transformed as the inverse of the cumulative standardized normal distribution (normsinv function in Excel) vs. either the concentration of alcohols expressed as a percentage or as the log-transformed percent. The best fit, based upon assessments of the residuals plots, was the inverse cumulative normal distribution-transformed proportion mortality vs. non-transformed percent alcohol. For the comparison of responses of Che and Cps to ethanol exposure, the percentage germination of spores was first normalized to 100% based upon the highest germination recorded for each species and trial. Following normalization, one percent was added to zero values and subtracted from 100% values to permit these data to be included in the regression analysis. These transformed data were subjected to multiple linear regression, which determined whether there were significant effects of alcohol concentration, duration of exposure, and responses by species.
Results and Discussion
Effect of sanitizers on Cps survival in 4 mm leaf disks.
Cps in leaf disks was killed (as determined by the inability to recover the fungus) in heavily infected small (4mm-d) leaf disks between 10 – 12 min by 70% ethanol and in 3 hr by 0.525% NaOCl (Fig. 1). Chlorophenol reduced survival but did not completely kill the Cps from the leaf tissue. There were significant time-dependent responses for ethanol (T = -3.23, P = 0.017, R2 = 0.63), NaOCl (T = -9.03, P < 0.01, R2 = 0.96), and for the lower concentration of chlorophenol (T = -5.51, P = 0.012, R2 = 0.91). The response to chlorophenol at 43 ppm did not fit a linear model (T = -2.33, P = 0.10, R2 = 0.64): there was reduced survival of Cps in leaf disks with exposure of 60 min or more (Fisher’s Exact test, P ≤ 0.01), but some Cps remained viable at all tested exposure durations.
Effect of sanitizers on Cps microsclerotia.
Individual ms were killed by 70% ethanol by 4 min, as evident from their inability to produce hyphae or conidia. In contrast, ms exposed to 0.525% NaOCl or 0.05% chlorophenol for 30 min retained viability (Table 1).
Effect of sanitizers against Cps conidia.
Exposure of conidia for 30 sec to the disinfestants effectively eliminated viability of conidia; however, dilution reduced the efficacy for alcohol and chlorophenol (Table 2). Multiple regression analysis of (cumulative normal inverse) transformed proportion mortality data of conidia exposed for 2 – 16 sec to 10 – 50% alcohol provided a good fit to a linear statistical model (R2 = 0.87) (Statistix 9, Analytical Software 2105 Miller Landing Rd, Tallahassee, FL 32312). This experiment found no statistical differences between ethanol and isopropanol (T = 0.89, P = 0.38). Within this range of exposure duration there were no significant increases in mortality associated with longer exposure (T = 1.57, P = 0.13), which suggests that wetting the conidia with an effective dose can be sufficient to result in mortality (Fig. 2). The only significant regression variable was alcohol concentration (P < 0.0001). From this experiment, mortality of 99% was predicted to result from exposure to 63.6% alcohol; 11 percent of spores did not germinate in the water check.
Citation: Journal of Environmental Horticulture 43, 2; 10.24266/0738-2898-43.2.83
Effect of ethanol and isopropanol on germination of conidia of Cps and Che.
Multiple linear regression analysis of transformed data revealed that conidia germination was strongly influenced by ethanol concentration and duration of exposure (Fig. 3, P < 0.0001 for each factor). There were no statistically significant differences observed between the two species (P = 0.11). For the exposure of three Cps isolates and 3 Che isolates to 70% isopropanol, even five sec of exposure was sufficient to kill all spores. No statistical analysis was done because germination was only seen with no exposure to isopropanol. For both 70% ethanol and 70% isopropanol, exposed conidia of Cps and Che died in about 5 sec.
Citation: Journal of Environmental Horticulture 43, 2; 10.24266/0738-2898-43.2.83
The dose-response for mortality of conidia versus alcohol concentration is unusual, because the best regression model was not based upon the log of the concentration but rather the non-transformed concentration. When this is considered along with previous evidence that there is an optimum concentration of about 70% ethanol for disinfestation (Kruse et al. 1963), indicating that this is a non-linear response, use of alcohols for disinfestation involves an unconventional receptor-poison interaction. These patterns may point towards solubilization of essential membrane components as the mechanism of mortality by alcohols, in which the polarity of the mixture of water and alcohol needs to match that of the target(s) for optimal effect.
We found some disagreement between the time-dependent response of conidia to alcohol exposure. One bioassay method involving exposure from 2 – 16 sec (Fig. 2) found no evidence for a time related response, whereas the other, with similar elapsed times measured for exposure, detected a highly significant effect of exposure duration (Fig. 3). The consequential finding from both studies is that 70% alcohol (either ethanol or isopropanol) completely disinfested conidia with about 5 sec of exposure.
Ethanol and isopropanol are both inexpensive compounds that are quick-acting as sterilants, and which evaporate and leave no residue. They are not corrosive and are generally considered nontoxic for skin exposure. Rutala et al. (2008) mention some shortcomings: alcohols can damage shellac coatings and tend to swell and harden rubber after prolonged and repeated use, and alcohols are flammable and consequently must be stored in a cool, well-ventilated area. They also evaporate rapidly, making extended exposure time difficult to achieve unless the items are immersed. Rushdy and Othman (2011) considered ethanol ineffective against bacterial biofilms and Turner et al. (2004) confirmed that isopropyl alcohol could be absorbed through intact skin.
Ethanol has long been considered to be more effective at 70% than at higher dilutions with water. Morton (1950) was concerned with the use of 95% ethanol as a skin disinfestant despite numerous citations saying that 95% ethanol was not as effective as 70% ethanol. He noted that the older publications (many enumerated by Harrington and Walker [1903]) tested efficacy using dried bacteria and Morton demonstrated that higher concentrations of ethanol were effective against moist bacteria. Kruse et al. (1963) also noted the conflicting reports about ethanol concentration but found 70% ethanol to be optimum for eradicating the vegetative phases of fungi air-dried on surfaces. Our results suggest that concentrations above 40% ethanol will be effective within 10 or 15 sec.
To compare the efficacy of isopropanol and ethanol, Smith (1947) performed tests on both dry and wet tubercle bacilli. In liquid suspensions, the organisms were killed in 30 sec by absolute and by 95% alcohol and in 1 min by 70% alcohol; 30% alcohol was ineffective. In dried smears, the tubercle bacilli were killed in 0.5 – 5 min by 50% alcohol and in 0.5 – 10 min by 70% alcohol. 95% alcohol was relatively ineffective. The action of isopropyl alcohol on dried bacilli paralleled that of ethyl alcohol except that the former was more active in lower concentrations. The effective range for isopropyl alcohol was 30 – 80%. Smith concluded that alcohol was suitable for skin disinfection at a concentration of 95% and for dry surfaces 70% ethyl or isopropyl alcohol was recommended.
There is some discussion of disinfesting tools and equipment in the plant pathology literature. Thomas (2007) isolated five bacterial clones from the spent alcohol used for tool-disinfestation while subculturing apparently clean, micropropagated, triploid watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai] cultures that harbored bacteria, while non-spore forming controls were killed within a few minutes. He considered this alcohol tolerance property of Bacillus spores to be a threat wherever alcohol is used as a surface disinfestant. James et al. (2012) evaluated nine disinfestants in vitro and in vivo for use against Phytophthora ramorum Werres, De Cock, & Man in't Veld mycelium and sporangia: Chemprocide® (a quaternary ammonium chloride product), PerCept™ (hydrogen peroxide) and 15% bleach treatments were effective for preventing P. ramorum recovery from contaminated plastic plant saucers and metal surfaces. Ethanol treatments for 5 min were among the least effective but significant improvements were observed with 10 min treatments of both 70% and 95% ethanol. Ogawa and Lyda (1960) examined the effect of alcohols on spores of Sclerotinia fructicola (G. Winter) Rehm, Rhizopus stolonifer (Ehrenb.) Vuill., and Gilbertella persicaria (E.D. Eddy) Hesselt. immersed for 1 min at 20 C, and found propanol most toxic, followed by isopropanol, ethanol, and methanol. Fifty percent ethanol could inactivate Sclerotinia conidia in 0.08 min on peach fruit surfaces and in 2 min on the flesh. Sixty per cent ethanol killed Gilbertella or Rhizopus sporangiospores on uninjured surfaces of peach fruits in 1 min, but on injured surfaces, 70% ethanol for 40 min was needed to kill Gilbertella and 70% for 60 min to kill Rhizopus.
Kodati et al. (2022) demonstrated that Cps conidia moved in sticky clumps under dry conditions could survive at least 9 d at relative humidity levels from 15 - 100%. This observation helps explain landscaper observations of boxwood blight spread and development despite following recommendations for pruning under dry conditions. The short time periods required for alcohols to be effective against Cps or Che are especially important to reduce disease spread by pruning. We conclude that either alcohol would be useful to sterilize tools and equipment after contact with any life stage of Calonectria.
Effect of sanitizers on Cps survival in 4 mm leaf disks. Disks could be disinfected with 70% ethanol and 0.525% NaOCl but not with chlorophenol at either 5.37 or 43 ppm. Statistical details are given in the text.
Effect on germination of Cps conidia exposed to alcohols: data shown are the percentage of spores that did not germinate and are the mean of five replicates of 100 spores. Dotted regression line, ethanol; solid line, isopropanol. Eleven percent of spores did not germinate in the distilled water control. There are no significant differences between alcohols and for duration of exposure within concentrations tested, but concentration is statistically significant (T = 14.3, P < 0.0001).
The effect of different concentrations of ethanol on germination of conidia of Calonectria henricotiae or C. pseudonaviculata. Multiple linear regression on cumulative normal distribution inverse-transformed proportion spore germination data revealed highly significant exposure time and concentration effects (P < 0.0001) and no significant differences between species (P = 0.11). One percent germination was added to zero values and subracted from 100% germination to include these data in the regression. The same response surface generated by the regression model from combined data is displayed for both species.
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